Stress increases the risk of psychiatric disorders such as post-traumatic stress disorders and depression. Women are twice as likely to suffer from these disorders as men. We do not fully understand the neurobiology underlying the sex difference in these stress- related disorders. The hypothalamic neuropeptides orexins are important for attention and arousal and regulate responses to stress, depression- and anxiety-related behaviors. However, little is known about sex differences in the expression of orexins or in the physiological and behavioral processes that they regulate. In preliminary studies in adult rats, females exhibited higher orexin mRNA, activation of orexin cells, and orexin concentrations in the cerebrospinal fluid compared to males under non-stressed conditions. In response to repeated restraint, females showed impaired habituation of behavioral and hypothalamic-pituitary-adrenal (HPA) responses and deficits in the cortically-mediated attention set-shifting task, a test of cognitive flexibility, compared to males. Based on these preliminary data, the central hypothesis of this application is that elevations in the expression and activation of orexins in females underlie sex differences in habituation to stress and in attention-related cognitive function. Four aims are designed to test this hypothesis at levels of analysis ranging from whole cell electrophysiology to behavior. In Specific Aim 1, we will test sex differences in orexin functions at the single cell level by using whole cell electrophysiology to assess whether differences in excitatory or inhibitory inputs to orexin neurons contribute to the observed sex differences. In Specific Aim 2, we will test whether circulating estrogens regulate orexin expression and electrophysiological activity and/or whether they drive the prepro-orexin promoter. We demonstrate sex differences in habituation of the HPA axis and struggling behavior and in cognitive performance in the cortically-mediated attentional set shifting task. In Specific Aims 3 and 4, we will test whether the higher orexin expression in females leads to these disruptions by stimulating or inhibiting orexins using DREADDs. Using multiple levels of analyses, the proposed research will provide the first comprehensive analysis of how orexins regulate sex differences in habituation and cognitive consequences of stress. With a better understanding of an important neural substrate underlying sex differences in stress responses, we may be able to develop improved, sex-specific, targets for stress-related psychiatric illnesses.